This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protein phosphorylation is a major mechanism of post-translational protein modification used to control cellular signalling. A challenge in phosphoproteomics is to identify the direct substrates of each protein kinase. We have developed a chemical strategy for delivery of a bio-orthogonal affinity tag to the substrates of an individual protein kinase. The kinase of interest is engineered to transfer a phosphorothioate moiety to phosphoacceptor hydroxyl groups on direct substrates. In a second non-enzymatic step, the introduced phosphorothioate is alkylated with p-nitrobenzylmesylate (PNBM). Antibodies directed against the modified phosphorothioate epitope recognize these labelled substrates, but not alkylation products of other cellular nucleophiles. Immunoaffinity chromatography allows the purification of these substrates, and mass spectrometry provides an attractive method for their rapid identification.